| ObjectivesUsing network pharmacology methods,predict the active ingredients and their mechanisms of hepatitis from the traditional Chinese medicine Tripterygium wilfordii Hook.F.,and use molecular docking technology for preliminary verification;Concanavalin A(ConA)induced autoimmunity in mice Autoimmune hepatitis(AIH)model was adopted to explore the therapeutic effect and mechanism of triptolide on AIH in mice,and to provide more research data for the clinical application and in-depth development of Tripterygium wilfordii Hook.F..Material and MethodsNetwork pharmacology:Search and filter the active ingredients and targets of Tripterygium wilfordii Hook.F.based on the TCMSP database,and search for the disease targets in the GeneCards,PubMed and OMIM databases and standardize them through the Uniprot database.Submitted to the jvenn website to screen the shared targets of the two,construct a PPI(protein-protein interactions)network through the String database,and use R software to draw a histogram of the Degree value.Upload the shared target information to the DAVID database for GO and KEGG enrichment analysis and visual processing,and use Cytoscape software to construct a "drug-active ingredient-target-pathway-disease" network model.Select the top 5 compounds in the degree of the active ingredient-target network of Tripterygium wilfordii Hook.F.and target molecules with Degree?60 in the PPI network Dot molecules to use PYMOL,AutoDockTools and AutoDock Vina software for molecular docking.Animal experiment:Male Balb/c mice were used as the research object,and ConA(20mg/kg)was injected into the tail vein to establish an AIH mouse model.The experimental animals were randomly divided into normal control group,ConA model group,and triptolide treatment group(0.4mg/kg),with 10 animals each group.After 12 hours of ConA injection,the eyeballs were taken for blood testing and the liver was harvested.Serum was used for testing liver functional indicators(ALT,AST),HE staining of paraffin sections of liver tissue,immunohistochemical detection of liver tissue morphology changes,Real-Time PCR and liquid chip technology to detect the expression of cytokines to explore its mechanism of action.ResultsNetwork pharmacology:(1)Tripterygium wilfordii Hook.F.and hepatitis shared targets network construction results:5 high-connection zone compounds in Tripterygium wilfordii Hook.F.were found,followed by kaempferol,triptolide,?-sitosterol,nobiliptin,and Stigmasterol;The nodes with Degree?60 in the PPI network include:AKT1,TNF,TP53,VEGFA,JUN,CXCL8,STAT3,PTGS2.(2)The shared targets enrichment analysis results:After KEGG enrichment analysis,the top 20 key signaling pathways involve metabolism,apoptosis,inflammation and other related signaling pathways,including:PI3K-Akt signaling pathway,TNF signaling pathway and Toll-like receptor signaling pathway and so on.(3)Molecular docking results:The five main active components of Tripterygium wilfordii Hook.F.can form a variety of covalent bonds with the key target protein of hepatitis,and have good affinity.Animal experiments:(1)Triptolide can significantly inhibit ConA-induced AIH in mice.(2)Compared with the normal control group,both alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in the ConA model group increased;after treatment with triptolide,ALT and AST decreased compared with the ConA model group,but It is higher than the normal control group.(3)The results of HE staining of liver tissue showed that the liver tissue of the normal control group was evenly stained,the liver lobule structure was clear,the liver cords were arranged neatly,and the morphology of hepatocytes was intact,with no obvious abnormal changes;the liver tissue of the ConA model group was darkly stained,Hepatocytes are arranged disorderly,the boundaries are blurred,hepatic sinusoids are congested,hepatocytes are mainly swollen and a small amount of necrosis,and inflammatory cell infiltration occurs in local areas;the hepatocytes of the mice in the triptolide treatment group are neatly arranged and clearly demarcated.The degree of swelling and necrosis is reduced,and the infiltration of inflammatory cells is reduced.(4)The CD4 immunohistochemical results of liver tissue showed that a small number of cells in the liver tissue of the normal control group showed a brown-yellow positive reaction;a large number of positive reaction cells appeared in the ConA model group,mostly located around the central vein and liver injury areas.The normal control group increased significantly(P<0.05),and the positive reaction cells decreased significantly after triptolide treatment(P<0.05).(5)Detection of cytokine expression in liver tissue:Real-Time PCR technology was used to detect the expression of cytokine mRNA related to inflammatory response and CD4+T cell differentiation in mouse liver tissue.The results showed that:compared with normal control group,the mRNA expression levels of Th1,Th2 and Th17 inflammatory cytokines in the ConA model group were significantly up-regulated,and the mRNA expression levels of inflammatory cytokines were significantly down-regulated after triptolide treatment(P<0.05).(6)Liquid-phase chip technology detected the expression of inflammatory cytokines in the liver tissue and serum of mice at the protein level.The results showed that compared with the normal control group,the level of inflammatory cytokines in the ConA model group was significantly higher(P<0.05).After triptolide treatment,the level of inflammatory cytokines was significantly reduced(P<0.05).Conclusion1.Tripterygium wilfordii Hook.F.has a potential therapeutic effect on hepatitis.The main active ingredients are kaempferol,triptolide,?-sitosterol,nobiliptin,and Stigmasterol.The possible mechanism is related to targets such as AKT1,CXCL8 and PTGS2.Related to PI3K-Akt,TNF,Toll and other signal pathways;2.Triptolide has a good preventive effect on ConA-induced AIH in mice;3.Triptolide exerts a therapeutic effect on AIH,and its preliminary pharmacological mechanism is related to inhibiting the recruitment of CD4+T cells to the liver and inhibiting the expression of inflammatory cytokines;... |
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